Supplementary Experimental Procedure 1:
Isolation of polysomal RNA and total RNA
Approximately 0.3 g of frozen tissue was extracted with 600 μl of polysome
extraction buffer (200 mM Tris-HCl, pH 8.5, 50 mM KCl, 25 mM MgCl2, 100 mg/ml
heparin, 50 mg/ml cycloheximide, 400 U/ml RNasin (Promega, Madison, WI), 2%
polyoxyethylene 10 tridecyl ether, 1% deoxycholic acid). The resuspended mixture
was incubated on ice for 5 min and then spun at 15,000 g for 5 min at 4°C. A total of
500 μl of supernatant was loaded on a 10 ml continuous sucrose gradient (15–50%)
and spun at 210,000 g for 3.5 h at 4°C. The distribution of the nucleic acids was
examined
by a
UV254
absorbance
profile
(model
#UV-6,
ISCO).
For
polysome-bound RNA (mRNAPL), RNA from polysomal fractions (PL) was purified
with phenol/chloroform extraction, LiCl precipitated and dissolved in RNase-free
H2O for further analyses. For total RNA (mRNASS), tissues were directly extracted
and purified via phenol/chloroform extraction, LiCl precipitated and dissolved in
RNase-free H2O for further analyses. Twenty microliters of 1:5000 diluted spike-in
DAP mRNA was added before RNA extraction (GeneChip Eukaryotic Poly-A
RNAcontrol kit, Ambion, Foster City, CA). For the detection of changes in ribosome
occupancy, the expression of spike-in DAP was used as a control to normalize the
efficiency of the RNA purification, cDNA synthesis and PCR process. The RNA
quantities of the NP and PL fractions were summed and set as 100% for the
calculations of the relative percentages of each transcript in the PL fraction.
PL% was used as a calibration factor for ‘Per-Chip’ normalization as described
previously (Kawaguchi et al, 2004; Branco-Price et al, 2005). PL% is the percentage
of the polysomal RNA relative to the total RNA as calculated by the following
formula:
PL% = RNA quantity in PL / (RNA quantity in both NP and PL) × 100%.
Supplementary Experimental Procedure 2:
Microarray Analysis
Two biological replicates were performed for total or polysomal RNA isolated from
each treatment indicated. Total RNAs were labeled and hybridized to Agilent
Arabidopsis V4 Oligo 4X44K Microarray (Agilent, Santa Clara, CA) as suggested by
the manufacturer. One μg of total RNA was amplified by a Quick-Amp Labeling kit
(Agilent Technologies, USA) and labeled with Cy3 (CyDye, PerkinElmer, USA)
during the in vitro transcription process. CyDye-labled cRNA (0.825 μg) was
fragmented to an average size of about 50-100 nucleotides by incubation with
fragmentation buffer at 60 oC for 30 minutes. Correspondingly fragmented labeled
cRNA is then pooled and hybridized to Agilent Arabidopsis V4 Oligo 4×44K
Microarray (Agilent Technologies, USA) at 60 °C for 17 h. After washing and drying
by nitrogen gun blowing, microarrays are scanned with an Agilent microarray scanner
(Agilent Technologies, USA) at 535 nm for Cy3. Scanned images are analyzed by
Feature extraction 9.5.3 software (Agilent Technologies, USA), and normalization
software is used to quantify signal and background intensity for each feature.
Gene expression data were extracted and calculated by GeneSpring 12.5 (Agilent,
Santa Clara, CA) and analyzed as described by Liu et al. (2012) with the following
modifications. First, probes marked as ‘_s_’ or ‘_x_’, indicating they are less specific,
were removed. Second, probe sets had to be marked as ‘present’ or ‘marginal’ in all
32 data sets. Third, the average intensity had to be ≥ 50 in both biological replicates
from at least one of the time points examined. For calculating the changes in both
steady-state and polysome-bound RNA, the average intensity in all 32 chips was
scaled to 500. All measurements with intensity < 0.01 were raised to 0.01. For
per-chip normalization, 50th percentile for the chips of steady-state mRNA
measurement and the PL% for the chips of polysome-bound mRNAs were used,
respectively. For per-gene normalization, ratios of GSH-treated to control samples
belonging to the same biological experiment were calculated. The above
normalizations were performed with the use of GeneSpring 12.5. Two-class
Significance Analysis of Microarrays (SAM) was used for identifying genes with
significantly different expressions (Tusher et al., 2001). Only genes that passed the
criterion of a false discovery rate (FDR, P-values for the Benjamini and Hochberg
method) listed in Supplementary Experimental Procedure 2 were selected for further
studies. Genes with expression ratios of ≥ two-folds or ≤ 1/2-fold were considered
upregulated or downregulated, respectively. A k-means cluster analysis (GeneSpring
12.5; Agilent) was performed to group the differentially expressed genes as shown in
Figure S11. Parameters included the specification of 4 clusters and 200 iterations, and
Pearson’s correlation was used for similarity measurement. The data sets were
deposited in NCBI’s Gene Expression Omnibus (series accession number
[GSE67356]).
Functional classification was performed by using the DAVID functional
annotation clustering tool (http://david.abcc.ncifcrf.gov/home.jsp) (Huang et al.,
2009). The functional clusters enrichment analysis was calculated by comparing the
whole Arabidopsis genome, and the highest classification stringency was chosen for
clustering.
Supplementary Experimental Procedure 3:
A flowchart showing the detailed data analysis procedures. The numbers of genes
meeting each criterion are in parentheses.
Supplementary Experimental Procedure 4:
Quantification of proline (Pro) and ABA contents
Four-week-old Arabidopsis plants under normal growth conditions were used for
the measurement of Pro contents. Lyophilized samples of approximately 100 mg of
plant material were extracted in 2 mL of 3% aqueous sulfosalicylic acid. A total of
500 mL of filtrate was reacted with 2 mL of ninhydrin and 500 mL of glacial acetic
acid in a test tube for 1 h at 100°C, and the reaction was terminated in an ice bath. The
reaction mixture was extracted with 1 mL of toluene and mixed vigorously with a test
tube stirrer for 15 to 20 s. The chromophore containing toluene was aspirated from the
aqueous phase, warmed to room temperature, and the absorbance was read at 520 nm
using toluene for a blank. The Pro concentration was determined from a standard
curve and calculated on a fresh weight basis as follows: [(mg Pro mL21 3 mL
toluene)/ 115.5 mg mmol21]/[(g sample)/5] = mmol Pro g21 fresh weight material.
For ABA quantification, lyophilized samples of approximately 100 mg of whole
seedlings were homogenized with 1 mL of 80% acetone and 20% water (v/v)
containing 1% (v/v) acetic acid in a 2-mL round-bottom tube. The homogenate was
incubated for 48 h at 4°C in darkness at 200 rpm. A total of 300 mL of the supernatant
was dried under vacuum. Following resuspension in 1 mL of 50% methanol (v/v) and
0.1 M NH4H2PO4 by vortexing, samples were purified by polyvinylpolypyrrolidone
(Sigma-Aldrich) and a C18 column (Sep-Pak Vac 3-cc [500-mg] C18 Cartridges;
Waters). After washing with 4 mL of 20% methanol and 2% acetic acid, ABA was
eluted by 4 mL of 55% methanol and 2% acetic acid. One milliliter of eluate was
vacuum dried and resuspend in 200 mL of standard Tris-buffered saline buffer. ABA
in these extracts was quantified using the Phytodetek ABA ELISA kit (Agdia)
according to the manufacturer’s instructions. The assay principle uses the competitive
antibody-binding method to measure concentrations of ABA in plant extracts.